A Novel GH Family 20 β-N-acetylhexosaminidase With Both Chitosanase and Chitinase Activity From Aspergillus oryzae

Abstract

Aminooligosaccharides possess various biological activities and can exploit wide applications in food, pharmaceutical and cosmetic industries. Commercial aminooligosaccharides are often prepared by the hydrolysis of chitin and chitosan. In this study, a novel GH family 20 β-N-acetylhexosaminidases gene named AoNagase was cloned from Aspergillus oryzae and expressed in Pichia pastoris. The purified AoNagase had maximal activity at pH 5.5 and 65°C. It exhibited good pH stability in the range of pH 6.0-7.5 and at temperatures below 50°C. AoNagase was capable of hydrolyzing not only colloidal chitosan (508.26 U/mg) but also chitin (29.78 U/mg). The kinetic parameters (K _m and V _max ) of AoNagase were 1.51 mM, 1106.02 U/mg for chitosan and 0.41 mM, 40.31 U/mg for colloidal chitin. To our knowledge, AoNagase is the first GH family 20 β-N-acetylhexosaminidase capable of hydrolyzing both chitosan and chitin. AoNagase is an endo-type β-N-acetylhexosaminidases and can potentially be used for the manufacturing of aminooligosaccharides.

Keywords: aminooligosaccharide; chitin; chitosan; glycoside hydrolase family 20; β-N-acetylhexosaminidase.

FIGURE 1

Phylogenetic analysis of AoNagase with 67 proteins from GH family 20 in the CAZy databases. Neighbor-joining tree shows phylogenetic relationships between AoNagase and other GH family 20 members from Uniprot and Protein Date Bank entries. All sequences from GH family 20 were labeled with names and GenBank accession numbers. The underlines indicate the proteins with known 3D structure.

FIGURE 2

The agarose gel electrophoresis (1%) of the PCR products (A). The SDS-PAGE (12%) of the proteins during purification of the recombinant AoNagase expressed in P. pastoris (B); Lane 1: The crude enzyme in the supernatant of lysates; Lane 2: The purified enzyme, eluted by 350 mM–400 mM NaCl solution.

FIGURE 3

The biochemical characterization of AoNagase, optimal pH (A), pH stability (B), optimal temperature (C), thermostability (D) and the effects of metal ions on AoNagase activity with chitosan (E) and colloidal chitin (F) as substrat. The results were the average of 3 sets of parallel samples, and the standard deviation (SD) was taken as the error bar.

FIGURE 4

Hydrolytic process of AoNagase toward chitosan, COS (DP 2–6) and colloidal chitin by TLC. The substrates were chitosan (A) (GlcN)₂ (B) (GlcN)₃ (C) (GlcN)₄ (D) (GlcN)₅ (E) (GlcN)₆ (F) and colloidal chitin (G). Lane M (GlcN)_1–6; Lane M₁ (GlcNAc)_1–3.